The ability to manipulate the mammalian genome, and in particular, the ability to develop animals with specific genes altered or inactivated has been invaluable to the study of gene function. The capability to modify or inactivate a gene can lead to unexpected discoveries of a gene and/or mechanisms responsible for disease with similar manifestations in humans. These genetically engineered animals are also useful for testing drug treatments and developing gene therapy strategies. (See, e.g., Bradley A., 1993, Recent Prog. Horm. Res. 48:237-251).
Mouse mutants have provided an extremely useful source of knowledge of mammalian development, cellular biology, and physiology, and have provided models for human diseases. An example of a well-known animal having a mutated or “knock-out” gene includes mice carrying a specifically modified or disrupted form of a chloride-channel gene. These mice develop a disease closely resembling human cystic fibrosis. Other examples of mice that have proven to be particularly valuable include those with alterations of genes encoding lymphocyte-specific tyrosine kinase p56.sup.lck and Lyt-2, alpha.-Calcium Calmodulin kinase II gene, the C/EPB.alpha. gene, and the BAX gene. (See, e.g., Snowouwaert et al., 1992, Science 257:1083-1088; Dorin et al., 1992, Nature 359:211-215; U.S. Pat. No. 5,625,122; U.S. Pat. No. 5,530,178; Silva et al., 1992, Science, 257:201; Wang et al., 1995, Science, 269:1108; Knudsen et al., 1995, Science, 270:960).
Determining how a gene functions ultimately requires genetic analysis in vivo. The mouse, for example, is a proven model system for studying various aspects of in vivo genetic analysis and mammalian development. (See, e.g., Paigen K., 1995, Nature Med. 1:215-220). Understanding how mammalian genes function, including genes from humans, has relied heavily on gene targeting technologies. Gene targeting allows for the generation of mice with a specifically-altered genotype.
Genetically altering specifically-targeted DNA sequences within eukaryotic genomes relies on homologous recombination to replace normal gene sequences in a cell with modified exogenous sequences that introduce the desired mutation. Such targeted replacement of a DNA sequence occurs in only a small fraction of the treated cells, while the incoming DNA is subject most often to random integrations. (See, e.g., Bollag et al., 1989, Annu. Rev. Genet. 23:199-225). More particularly, exogenous sequences transferred into eukaryotic cells undergo homologous recombination with homologous endogenous sequences only at very low frequencies, and are so inefficiently recombined that large numbers of cells must be transfected, selected, and screened in order to generate a desired correctly targeted homologous recombinant. (See, e.g., Kucherlapati et al., 1984, Proc. Natl. Acad. Sci. (U.S.A.) 81: 3153; Smithies, O., 1985, Nature 317: 230; Song et al., 1987, Proc. Natl. Acad. Sci. (U.S.A.) 84: 6820; Doetschman et al., 1987, Nature 330: 576; Kim and Smithies, 1988, Nucleic Acids Res. 16: 8887; Shesely et al., 1991, Proc. Natl. Acad. Sci. (U.S.A.) 88: 4294; Kim et al., 1991, Gene 103: 227).
The most common approach to producing these transgenic animals involves the disruption of a target DNA sequence by insertion of a DNA construct encoding a selectable marker gene flanked by DNA sequences homologous to part of the target gene. When properly designed, the DNA construct effectively integrates into and disrupts the targeted gene via homologous recombination, thereby preventing the normal expression of an active gene product encoded by that gene.
Typically, gene targeting strategies employed to generate animals having specific mutations involve the following steps: 1) directed mutagenesis of the target gene in vitro; 2) introduction of the mutant gene into cultured embryonic stem cells; 3) screening for cell lines carrying the desired homologous recombination (i.e., gene replacement) event; and 4) generation of mice that transmit the mutant gene. (See, e.g., Capecchi, 1989, Trends In Genetics 5(3):70-76; Capecchi, 1989, Science 244(4910):1288-1292).
Directed mutagenesis of the target gene in vitro can be achieved using standard molecular biology and DNA cloning techniques. Typically, a functionally-relevant gene sequence is deleted and replaced with a selectable marker gene. The neo gene, which encodes neomycin phosphotransferase and confers cellular resistance to neomycin, G418 and related drugs, is routinely used as the selectable marker gene. In general, the deletion and replacement of the functionally-relevant gene are designed to generate a null mutation in the target gene disrupting its normal activity or function.
To introduce a mutant gene into cultured embryonic stem cells, a genetic construct or targeting vector is grown as a DNA plasmid in bacteria and then transfected into murine embryonic stem cells in vitro. The desired transfected cells, which represent a small fraction of the total cell population, are purified from those that failed to take in the vector by positively selecting for the marker gene in the transfected cells. Specifically, addition of neomycin to the culture kills untransfected cells, thus, selecting for the outgrowth of resistant transfected cells that express the neo gene. These resistant cells grow into colonies, each representing clonal populations derived from independently transfected cells.
Screening for cell lines carrying the desired homologous recombination event allows for the identification of cells in which the specific gene replacement has occurred. Given that random integration typically occurs more frequently than does homologous recombination, only a small minority of the colonies will be derived from cells having homologous gene replacement. This screening process requires that DNA samples isolated from individual cell lines be analyzed for homologous recombination, usually by the polymerase chain reaction (PCR) or DNA blot hybridization (Southern blotting).
To generate mice that transmit the mutant gene, embryonic stem cells carrying the desired homologous recombination event can be injected into mouse blastocysts. The blastocysts are then implanted into pseudopregnant females to generate chimeric mice, comprised of both mutant and wild-type cells. If the germline has been populated with mutant cells, then the targeted allele can be transmitted to subsequent generations, and the phenotypic consequences of the mutation can be assessed.
One of the most challenging aspects in generating animals comprising a targeted gene modification is the identification and isolation of the rare cell line that carries the homologous recombination event. One approach to combating this difficulty involves the addition of a negative selection step. This technique allows for the enrichment of the transfected cell population for the desired cells, relying on negative selection to specifically kill cells that carry random integrations. (See, e.g., U.S. Pat. No.: 5,627,059). In addition to the general techniques described above, this positive/negative selection (PNS) method requires the cloning of a negative selectable marker into the targeting vector and a further negative selection step. The gene encoding thymidine kinase (TK) is routinely used as the negative selection marker in the PNS method.
The PNS method involves a process in which a first drug is added to the cell population, for example, a neomycin-like drug to select for growth of transfected cells, i.e. positive selection. A second drug, such as FIAU is subsequently added to kill cells that express TK, i.e. negative selection. However, addition of the second drug can be quite toxic to the cells and may negatively affect the ability of the cells to populate the germline. (See, e.g., Yanagawa et al., 1999, Transgenic Research 215-221). Unfortunately, in addition to homologous recombination, many random integration events will also inactivate TK. Indeed, although the negative selection enriches the cell population for homologous recombinants, this population still predominantly contains random integration events.
Mammalian cells have a remarkable ability to support nonhomologous recombination of incoming DNA. For example, animals bearing a foreign gene randomly inserted into their genome to express a foreign protein are reported in the art. These animals are most often used to produce, for example, a pharmaceutical substance. Typically, in this process expression of the foreign gene's coding sequence is under the control of a promoter.
Previous studies demonstrated that control of eukaryotic transcriptional promoters, can be modified to respond to bacterial transcription factors. (See, e.g., Hu and Davidson, Molecular and Cellular Biology 10(12):6141-6151; Hu and Davidson, 1991, Gene 99(2):141-150; Hu and Davidson, 1987, Cell 48(4):555-566; Hu and Davidson, 1988, Gene 62(2):301-313; Hannan et al., 1993, Gene 130(2):233-239).
However, the method of expressing a foreign gene of interest in a mammalian cell by randomly inserting the gene into the genome of the animal is contrary to the process of gene targeting. Gene targeting relies on homologous recombination, wherein the goal is to produce an animal carrying a modified or disrupted form of a specific gene of interest.
As described above, the experimental challenge in gene targeting lies in identifying the rare colonies of cells carrying the desired mutated target gene. As it is often difficult to differentiate between random insertions and homologous recombination, a need in the art exists for methods that enhance and promote the recovery of homologous recombination events, while providing a faster, more efficient, and more reliable means for generating cells and animals having specific genes modified or disrupted.